Fig. 5.
Fig. 5. Direct activation of Elk1 by thrombin. / (A) Heterologous expression of the Elk1 transcription factor in primary fibroblasts and activation by thrombin. All transfections were performed as in Figure 1C except that the cells were cotransfected with 2 μg of a reporter construct containing 6 Gal4 binding elements in series immediately upstream of the luciferase gene (pFR-Luc) and with 2 μg of the expression vector for the fusion protein pFA-Gal4dbd-Elk1AD, which contains the Gal4 protein DNA binding domain (Gal4dbd) and the Elk1 protein activation domain (Elk1AD). Stimulation of the cotransfected cells with thrombin resulted in 16-fold increase over the control (iv). As controls, cells were transfected with 2 μg of pFR-Luc and 2 μg of pcDNA3.0 vector (i), or cotransfected with pFR-Luc and 2 μg of the expression vector for the Gal4dbd and then stimulated by thrombin (ii), or cotransfected with pFR-Luc and 2 μg of the pFA-Gal4dbd-Elk1AD in the absence of stimulation by thrombin (iii). (B) Overexpression of the Elk1 transcription factor and thrombin treatment on 9E3/cCAF expression. The transient transfections were performed as described in Figure 1C. Cotransfection of 2 μg of pCMV-Elk1 with 2 μg of p683 in primary fibroblasts resulted in a 2-fold increase in activation of the reporter gene (compare i and iii), whereas without Elk1 overexpression, thrombin stimulation enhanced transcription 3-fold (compare i and ii and with Figures 2 and 3, but in the latter note the scale differences). When the cotransfected cells were treated with thrombin, a synergistic effect was observed (iv). The transfections for conditions (i) and (ii) also included 2 μg of pcDNA3.0 vector. (C) Immunoblot analysis using an antibody specific for cCAF. Supernatants of fibroblasts treated as in panel B were resolved in 20% polyacrylamide-glycerol gel. The synergistic effect on transcription activation of 9E3/cCAF is reflected in an increase in the amount of cCAF protein produced by the fibroblasts. This figure shows representative experiments in all cases. Each experiment was performed at least twice, using different batches of cells. Bars in panels A and B represent SEMs of 3 samples/condition in the same batch of cells.

Direct activation of Elk1 by thrombin.

(A) Heterologous expression of the Elk1 transcription factor in primary fibroblasts and activation by thrombin. All transfections were performed as in Figure 1C except that the cells were cotransfected with 2 μg of a reporter construct containing 6 Gal4 binding elements in series immediately upstream of the luciferase gene (pFR-Luc) and with 2 μg of the expression vector for the fusion protein pFA-Gal4dbd-Elk1AD, which contains the Gal4 protein DNA binding domain (Gal4dbd) and the Elk1 protein activation domain (Elk1AD). Stimulation of the cotransfected cells with thrombin resulted in 16-fold increase over the control (iv). As controls, cells were transfected with 2 μg of pFR-Luc and 2 μg of pcDNA3.0 vector (i), or cotransfected with pFR-Luc and 2 μg of the expression vector for the Gal4dbd and then stimulated by thrombin (ii), or cotransfected with pFR-Luc and 2 μg of the pFA-Gal4dbd-Elk1AD in the absence of stimulation by thrombin (iii). (B) Overexpression of the Elk1 transcription factor and thrombin treatment on 9E3/cCAF expression. The transient transfections were performed as described in Figure 1C. Cotransfection of 2 μg of pCMV-Elk1 with 2 μg of p683 in primary fibroblasts resulted in a 2-fold increase in activation of the reporter gene (compare i and iii), whereas without Elk1 overexpression, thrombin stimulation enhanced transcription 3-fold (compare i and ii and with Figures 2 and 3, but in the latter note the scale differences). When the cotransfected cells were treated with thrombin, a synergistic effect was observed (iv). The transfections for conditions (i) and (ii) also included 2 μg of pcDNA3.0 vector. (C) Immunoblot analysis using an antibody specific for cCAF. Supernatants of fibroblasts treated as in panel B were resolved in 20% polyacrylamide-glycerol gel. The synergistic effect on transcription activation of 9E3/cCAF is reflected in an increase in the amount of cCAF protein produced by the fibroblasts. This figure shows representative experiments in all cases. Each experiment was performed at least twice, using different batches of cells. Bars in panels A and B represent SEMs of 3 samples/condition in the same batch of cells.

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